Biochemical and candidate gene approaches over the past four decades have led to the identification of molecules that function to regulate excitatory, glutamatergic synapse formation and synaptic transmission. In contrast, far less is known about inhibitory, GABAergic synapse formation and function. It has previously been determined that knockdown of the transmembrane class 4 Semaphorin Sema4D in the postsynaptic neuron leads to a decrease in the density of GABAergic synapses formed onto that neuron, without an effect on glutamatergic synapse density (Paradis et al., “An RNAi-Based Approach Identifies Molecules Required for Glutamatergic and GABAergic Synapse Development,” Neuron., 53:217-232 (2007)). Further, immunohistochemical analysis of hippocampi isolated from mice in which the Sema4D gene was constitutively deleted (Shi et al., “The Class IV Semaphorin CD100 Plays Nonredundant Roles in the Immune System: Defective B and T Cell Activation in CD100-Deficient Mice,” Immunity, 13:633-642 (2000)) revealed a decrease in intensity of GABA-synthesizing enzyme GAD67 immunoreactivity in the neuropil (Paradis et al., “An RNAi-Based Approach Identifies Molecules Required for Glutamatergic and GABAergic Synapse Development,” Neuron., 53:217-232 (2007)). This result is consistent with a deficit in GABAergic synapse development in the absence of Sema4D in vivo (Paradis et al., “An RNAi-Based Approach Identifies Molecules Required for Glutamatergic and GABAergic Synapse Development,” Neuron., 53:217-232 (2007)). Thus, these experiments identify Sema4D as one of only a few molecules described thus far that preferentially regulate GABAergic synapse formation.
The mammalian Semaphorin family of proteins consists of 20 secreted and membrane-bound molecules grouped into five different classes based on their sequence homology and protein domain structures (Tran et al., “Semaphorin Regulation of Cellular Morphology,” Ann. Rev. Cell Dev. Biol., 23:263-292 (2007); Yazdani et al., “The Semaphorins,” Genome Biol., 7:211 (2006); Zhou et al., “Semaphorin Signaling: Progress Made and Promises Ahead,” Trends Biochem. Sci., 33:161-170 (2008)). Sema4D signaling is required for the proper development and function of a variety of organ systems including the immune system, cardiovascular system, and CNS (Ch'ng et al., “Roles of Sema4D and Plexin-B1 in Tumor Progression,” Mol. Cancer, 9:251 (2010); Kruger et al., “Semaphorins Command Cells to Move,” Nat. Rev. Mol. Cell Biol., 6:789-800 (2005); Pasterkamp et al., “Semaphorin Function in Neural Plasticity and Disease,” Curr. Opin. Neurobiol., 19:263-274 (2009); Takamatsu et al., “Diverse Roles for Semaphorin-Plexin Signaling in the Immune System,” Trend Immunol., 33:127-135 (2012); Yazdani et al., “The Semaphorins,” Genome Biol., 7:211 (2006)). The hallmark of a Semaphorin family member is the extracellular Semaphorin (Sema) domain: a conserved, cysteine-rich region of about 500 amino acids at the N-terminus of the protein (Yazdani et al., “The Semaphorins,” Genome Biol., 7:211 (2006)). Sema4D is a transmembrane protein with a short intracellular domain in addition to its extracellular Sema domain. Currently, identifiable protein motifs in the intracellular domain of Sema4D have not been described and a function for this region has yet to be determined (Ch'ng et al., “Roles of Sema4D and Plexin-B1 in Tumor Progression,” Mol. Cancer, 9:251 (2010); Pasterkamp et al., “Semaphorin Function in Neural Plasticity and Disease,” Curr. Opin. Neurobiol., 19:263-274 (2009); Takamatsu et al., “Diverse Roles for Semaphorin-Plexin Signaling in the Immune System,” Trend Immunol., 33:127-135 (2012)). Therefore, all of the biological functions ascribed thus far to Sema4D can be attributed to the extracellular region, containing the conserved Sema domain through which Sema4D binds to its putative receptors: PlexinB family members and CD72 (Takamatsu et al., “Diverse Roles for Semaphorin-Plexin Signaling in the Immune System,” Trend Immunol., 33:127-135 (2012); Tamagnone et al., “Plexins are a Large Family of Receptors for Transmembrane, Secreted, and GPI-Anchored Semaphorins in Vertebrates,” Cell, 99:71-80 (1999); Yazdani et al., “The Semaphorins,” Genome Biol., 7:211 (2006)).
Recently, time-lapse imaging studies over the course of several hours have provided some insight into the cell biology of GABAergic synapse development (Dobie et al., “Inhibitory Synapse Dynamics: Coordinated Presynaptic and Postsynaptic Mobility and the Major Contribution of Recycled Vesicles to New Synapse Formation,” J. Neurosci., 31:10481-10493 (2011); Wierenga et al., “GABAergic Synapses are Formed Without the Involvement of Dendritic Protrusions,” Nat. Neurosci., 11:1044-1052 (2008)). For example, live-imaging of GABAergic synapse formation in hippocampal slices revealed that, in contrast to glutamatergic synapse development, GABAergic synapses form at pre-existing axodendritic crossings without the involvement of axonal or dendritic protrusions (Wierenga et al., “GABAergic Synapses are Formed Without the Involvement of Dendritic Protrusions,” Nat. Neurosci., 11:1044-1052 (2008)). In addition, time-lapse imaging in maturing neuronal cultures of labeled components of GABAergic synapses, such as GABAA receptors and Gephyrin, has revealed that synaptic components are transported in mobile packets to synaptic sites along dendrites (Dobie et al., “Inhibitory Synapse Dynamics: Coordinated Presynaptic and Postsynaptic Mobility and the Major Contribution of Recycled Vesicles to New Synapse Formation,” J. Neurosci., 31:10481-10493 (2011); Maas et al., “Neuronal Cotransport of Glycine Receptor and the Scaffold Protein Gephyrin,” J. Cell Biol., 172:441-451 (2006); Twelvetrees et al., “Delivery of GABAARs to Synapses is Mediated by HAP1-KIF5 and Disrupted by Mutant Huntingtin,” Neuron, 65:53-65 (2010)). However, these previous studies have not addressed either the mechanism or time frame of assembly of GABAergic synapses in response to a specific synaptogenic signal.
While the underlying cause of epileptogenesis remains largely unknown, the major phenotype of epileptic seizures is an increase in neuronal activity either in a specific focal region or globally (Morimoto et al., “Kindling and Status Epilepticus Models of Epilepsy: Rewiring the Brain,” Prog. Neurobiol., 73:1-60 (2004)). Studies of temporal lobe epilepsy reveal an increase in reorganization of neuronal connections including increased excitatory axon sprouting and synaptogenesis (Morimoto et al., “Kindling and Status Epilepticus Models of Epilepsy: Rewiring the Brain,” Prog. Neurobiol., 73:1-60 (2004)). Additionally, animal studies suggest that seizure activity can be facilitated by a loss of inhibitory control on neighboring neurons (Cossart et al., “Dendritic but not Somatic GABAergic Inhibition is Decreased in Experimental Epilepsy,” Nat. Neurosci., 4:52-62 (2001); Depaulis et al., “Quiescence and Hyporeactivity Evoked by Activation of Cell Bodies in the Ventrolateral Midbrain Periaqueductal Gray of the Rat,” Exp. Brain Res., 99:75-83 (1994); Kobayashi et al., “Reduced Inhibition of Dentate Granule Cells in a Model of Temporal Lobe Epilepsy,” J. Neurosci., 23:2440-2452 (2003)), illustrating the importance of precise regulation of inhibition and excitation within neural networks. However, the molecular mechanisms and time course of the development of inhibition in neuronal circuits are not well understood and biological targets affecting such inhibitory synapses, as well as agents for modulating such targets, are not known. Thus, there is a great need in the art to elucidate such molecular mechanisms, biological targets, and agents for treating, diagnosing, prognosing, and preventing neurological disorders that could benefit from increasing inhibitory synapse formation and activity.
The present invention is directed to overcoming these and other deficiencies in the art.